Seiichi Tsukamoto

Fatty Acids Bound to Recombinant Tear Lipocalin and Their Role in Structural Stabilization

A variant of human tear lipocalin was expressed in Escherichia coli, and the bound fatty acids were analysed by gas chromatography, mass spectroscopy and nuclear magnetic resonance spectroscopy. Five major fatty acids were identified as hexadecanoic acid (palmitic acid, PA), cis-9-hexadecenoic acid (palmitoleic acid), 9,10-methylenehexadecanoic acid, cis-11-octadecenoic acid (vaccenic acid) and 11,12-methyleneoctadecanoic acid (lactobacillic acid). The composition of the bound fatty acids was similar to the fatty acid composition of E. coli extract, suggesting that the binding affinities are similar for these fatty acids. The urea-induced and thermal-unfolding transitions of the holoprotein (nondelipidated), apoprotein (delipidated) and PA-bound protein were observed by circular dichroism. Holoproteins and PA-bound proteins showed the same stability against urea and heat, and were more stable than apoprotein. These results show that each bound fatty acid stabilizes recombinant tear lipocalin to a similar extent.

Non‐native α‐helix formation is not necessary for folding of lipocalin: Comparison of burst‐phase folding between tear lipocalin and β‐lactoglobulin

Tear lipocalin and β‐lactoglobulin are members of the lipocalin superfamily. They have similar tertiary structures but unusually low overall sequence similarity. Non‐native helical structures are formed during the early stage of β‐lactoglobulin folding. To address whether the non‐native helix formation is found in the folding of other lipocalin superfamily proteins, the folding kinetics of a tear lipocalin variant were investigated by stopped‐flow methods measuring the time‐dependent changes in circular dichroism (CD) spectrum and small‐angle X‐ray scattering (SAXS). CD spectrum showed that extensive secondary structures are not formed during a burst‐phase (within a measurement dead time). The SAXS data showed that the radius of gyration becomes much smaller than in the unfolded state during the burst‐phase, indicating that the molecule is collapsed during an early stage of folding. Therefore, non‐native helix formation is not general for folding of all lipocalin family members. The non‐native helix content in the burst‐phase folding appears to depend on helical propensities of the amino acid sequence. Proteins 2009.

Relationship between chain collapse and secondary structure formation in a partially folded protein

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil-globule transition)? To answer this question, we measured small-angle X-ray scattering for a series of β-lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil-globule transitions.

Chloride-ion concentration dependence of molecular dimension in the acid-denatured state of equine β-lactoglobulin

The chloride-ion concentration dependence of the molecular dimension in the acid-denatured state of equine β-lactoglobulin (ELG) was investigated by small-angle X-ray scattering. In the presence of chloride ion, ELG has a globular and compact conformation (the A state). The molecular dimension of ELG increases little with decreasing chloride-ion concentration. A remarkable dependence was observed for a mutant protein in which both Cys66 and Cys160 were replaced with Ala (C66A/C160A). In the presence of chloride ion, C66A/C160A has a globular and compact conformation, like the wild type. In the absence of chloride ion, however, the molecular dimension and shape was close to that in the urea-unfolded state. Previously, we have shown that the helix content in the acid-denatured state increases with decreasing chloride-ion concentration [Yamada et al. (2006). Proteins Struct. Funct. Bioinf. 63, 595–602]. These results suggest that the secondary structure in the A state is mainly determined by non-local interactions. When they are absent in an expanded conformation, the local interactions become predominant and the amount of non-native α-helix increases.

Delineation of Solution Burst-Phase Protein Folding Events by Encapsulating the Proteins in Silica Gels

Many studies have shown that during the early stages of the folding of a protein, chain collapse and secondary structure formation lead to a partially folded intermediate. Thus, direct observation of these early folding events is crucial if we are to understand protein-folding mechanisms. Notably, these events usually manifest as the initial unresolvable signals, denoted the burst phase, when monitored during conventional mixing experiments. However, folding events can be substantially slowed by first trapping a protein within a silica gel with a large water content, in which the trapped native state retains its solution conformation. In this study, we monitored the early folding events involving secondary structure formation of five globular proteins, horse heart cytochrome c, equine β-lactoglobulin, human tear lipocalin, bovine α-lactalbumin, and hen egg lysozyme, in silica gels containing 80% (w/w) water by CD spectroscopy. The folding rates decreased for each of the proteins, which allowed for direct observation of the initial folding transitions, equivalent to the solution burst phase. The formation of each initial intermediate state exhibited single exponential kinetics and Arrhenius activation energies of 14-31 kJ/mol.

Effect of non-native helix destabilization on the folding of equine β-lactoglobulin

β-lactoglobulin forms a non-native α-helix during an early stage of folding. To address the role of the non-native structure in the folding process, we designed several mutants of equine β-lactoglobulin with reduced helical propensity in the non-native helix region. One of them, A123T, showed a similar structure to that of the wild-type protein; its folding kinetics was investigated by stopped-flow circular dichroism (CD) and fluorescence. Although A123T showed a reduced burst-phase CD intensity, its folding rate was similar to that of the wild-type protein, which indicated that the formation of the non-native helix does not accelerate or decelerate the folding reaction.